The invention relates to the field of protein purification and the recovery of large proteinaceous material through small, nanometer sized, pore exclusion filters for removal of contaminants such as viral pathogens. The invention relates to the use of additives to promote solubility of proteins in solutions being filtered for the purpose of removing pathogens, particularly viral pathogens, and has particular applicability to the purification of large proteinaceous biomolecules such as immunoglobulins.
Liquid and gas separation processes are well known in the art. Most common separation processes involve a phase change, which increases the cost of the processes and often requires excessive temperature changes which can alter the product. Membrane separations, however, can achieve desired levels of separation without a change in the substances"" phase. In essence, membrane separation selectively forces one or more substances through pores of a filter, leaving one or more larger substances behind. This process is often repeated with diminishing filter pore sizes until a satisfactory level of separation is achieved.
The use of nanofiltration to remove contaminants such as virus particles from parenteral protein products is based upon the ability of a filter of defined pore size to allow a soluble protein to pass through while denying passage of the larger viral particles (DiLeo, A J, et al, BioTechnology 1992, 10: 182,188.) Removal of virus from large biomolecules such as immunoglobulins (monoclonal or polyclonal antibodies), by size exclusion, is hindered by the difficulty of passing the large biomolecules through pore sizes of nanometer size, typically 12-15 nm. While a protein in solution, even one as large as an immunoglobulin, is expected to have a molecular radius much smaller than a viral particle, several factors can lead to an effective reduction in pore size and sieving coefficient. Some of these factors are due to interactions between the protein and the filter surface resulting in build up on the membrane surface known as a gelation or polarization layer. Other factors, such as protein self-association or aggregation, cause the protein to be trapped by the filter due to formation of masses too large to pass through the filter pores or that have surface characteristics that exhibit affinity for the membrane surface or pore surfaces causing them to adhere to the membrane instead of passing through.
International patent application, WO 9600237, describes methods for successful nanofiltration using pore sizes as small as 15 nm to filter purified proteins of molecular weight less than 150 kDa. WO 9600237 discloses the use of salt concentrations lying in the range from about 0.2 M up to saturation of the solution in virus-filtering of proteins, polysaccharides, and polypeptides to increase sieving coefficients. The advantage of the salt is stated by the applicants to be because the xe2x80x9cprotein contractsxe2x80x9d and more easily passes through the filter. The use of a high salt content according to this method is also suggested to enable the use of xe2x80x9cdead-endxe2x80x9d filtering with membranes having pore sizes of 5-30 nm. Dead-end filtering refers to the practice of using a single pump to force fluid through the membrane from the surface. Dead-end filtration is simpler and more cost effective than tangential filtering process wherein a first pump maintains constant flow rate at the surface of the membrane and a second pump draws the protein through the filter by creating a negative pressure (suction) at the back of the membrane.
U.S. Pat. No. 6,096,872 recognized the utility of adding surfactants along with high ionic strength buffering during nanofiltration to remove viruses from immunoglobulin containing solutions in order to reduce protein dimerization, trimerization and aggregation, the teachings of which are hereby incorporated herein by reference.
It is also generally known that in order to reduce the interaction of a substance with the membrane surface, the xe2x80x9czeta-xe2x80x9d or xe2x80x9cz-xe2x80x9dpotential of the membrane surface should not be electrically attractive to that substance and altering the charge properties of the membrane can minimize surface precipitation. For example, U.S. Pat. No. 6,177,011 teaches that the neutralization of surface charges measured as zeta potential can reduce surface adsorption of membrane-fouling substances during reverse osmosis filtration processes where the substance carries a charged group. Changes in pH and salt concentration are other means of altering the z-potential of both the solutes and the membrane surface. In some cases, however, the manipulation of the z-potential by the addition of salt is counter-productive, resulting in an increase in soluble aggregation and an increase in the hydrophobic character of the membrane surface which may promote interaction with hydrophobic protein regions. Pall, et al (Colloids and Surfaces 1 (1980), 235-256.), reported that the phenomenon of removal of particles smaller than the pores of a filter is due to adherence of the particles to the pore walls under conditions wherein the particles and the pore walls are oppositely charged or alternatively wherein the zeta potential of the particles and the pore walls of the membrane are both low. Zierdt (Applied and Environmental Microbiology, (1979) 38:1166-1172) attributed the aforementioned phenomenon to electrostatic forces. Furthermore, these modifications do not address the effects of molecular geometry or protein aggregation in solution on membrane filtration.
In addition to the considerations of buffer components and their concentrations, care must be take to maintain the protein to be filtered in a concentration appropriate to maintaining good flow and minimal transmembrane pressure across the filter. WO 9837086 teaches the addition of buffer to the retentate in order to maintain transmembrane pressure during tangential flow of a pretreatment step to remove proteins having a molecular weight greater than that of the product protein(s). WO 9837086 further notes that nanofiltration is limited to therapeutic proteins having a molecular weight up to 150 kDa. Immunoglobulin G molecules are composed of two heavy chains and two light chain polypetides all covalently linked and have an average molecular weight of about 180 kDa. U.S. Pat. No. 6,096,872 seeks to address the problem of how to filter viruses from IgG products by including a non-ionic excipient with relatively high (physiological which is about 300 mOsm) ionic strength buffer. The use of high ionic strength buffers, however, may lead to protein aggregation or create the problem of salt removal from the product formulation. U.S. Pat. No. 6,096,872 teaches and claims a second nanofiltration step to concentrate the immunoglobulin and collect it in a low ionic strength buffer.
These methods suffer from various disadvantages, particularly in their efficiency. It is therefore the object of the present invention to overcome the short-comings of the prior art, particularly in developing a system for efficiently filtering pathogenic viruses from immunoglobulin products, thereby providing virally cleared, pure immunoglobulin for injection.
The molecular configuration or size of a protein species has been predicted by changes in the partial specific volume and self-association of proteins. The change in partial specific volume of proteins so modified has been demonstrated by the independent measurements of sedimentation coefficients using analytical centrifugation. The method described herein uses the addition of a clathrate modifying substance to modify the molecular configuration of the protein to minimize specific volume and aggregation thereby enhancing passage of the protein through the membrane in a nanofiltration process.
The method of the invention maximizes protein passage during membrane filtration by using buffer additives aimed to increase the hydrophobicity of the membrane surface and decrease the hydrodynamic radius of the protein as well as reduce the tendency for the self-association of the protein desired to be filtered. The method of the invention first maximizes protein passage by decreasing the pH and the salt of the buffer which increases the hydrophobicity of the membrane surface and decreases the hydrodynamic radius of the protein. Secondly, a clathrate modifer is included in the buffer which modifier decreases the hydrodynamic radius of the protein while minimizing the tendencies for the protein to associate with either itself or the membrane filter. Thirdly, the process optionally includes continuous in-line monitoring of the filtration in order to maintain the above parameters of pH and clathrate modifier constant while maintaining low local levels of soluble protein. The use of the methods of the invention result in an increase in sieving coefficient and the ability to maintain reduced trans-membrane pressure during virus particle filtration. The process is applicable to the purification of any large proteinaceous biomolecule, particularly immunoglobulins. The immunoglobulins may be a monoclonal or polyclonal immunoglobulin.
The clathrate modifier is perferably a polyol sugar or sugar alcohol having from 4 to 8 hydroxyl groups. Examples of preferred polyols are sugars, including mono-saccharides and disaccharides preferably sucrose. The concentration of the polyol used as a clathrate modifier will generally be 5% w/v or greater. The use of sucrose causes a decrease in the size of the molecule and a reduction in the tendency for self-association of the protein desired to be freed from virus particles.
Thus, the invention contemplates a method for purifying a proteinacious material such as an immunoglobulin comprising the steps of:
(a) admixing the proteinaceous material with:
(i) a low pH, low conductivity buffer solution formulated to reduce the pH between 5.0 and 6.0, and to achieve an ionic strength of less than 30 mS/cm;
(ii) a non-ionic surfactant; and
(iii) a clathrate modifier;
(b) performing nanofiltration on the proteinaceous material to obtain a purified material substantially free of viral particles.
Preferably, the clathrate modifier is a polyol sugar or sugar alcohol having from 4 to 8 hydroxyl groups.
The method of the invention may also include conducting an in-line pre-filtering step and monitoring the concentration of the material by installing an in-line concentration controlling monitor to maintain the parameters of pH, and protein concentration within pre-set ranges optimal for the material being purified.